The kidney collecting duct is the final site for the regulation of proton secretion required to maintain hydrogen ion balance. Hydrogen ion transport in this segment is carried out by The intercalated cells carry out ion transport in this segment by means of a vacuolar-type H+ATPase that is greatly amplified and distributed in a polarized distribution on the plasma membrane. The applicant recently demonstrated that one isoform of the 56 kD subunit of the vacuolar H+ATPase exhibits greatly amplified expression in the renal intercalated cells, but is present at low or undetectable levels in other tissues. It is the aim of this project to examine the mechanisms regulating expression of the gene encoding the "kidney" isoform of the vacuolar H+ATPase 56 kD subunit, in order to attain insights into the amplification and specialized use of the vacuolar H+ATPase that occurs in proton transporting cells. The specific aims are: 1) Clone the gene and determine the gene structure of the 5' flanking region and the first intron and exon of the rat "kidney" Mr 56,000 subunit of the vacuolar H+ATPase. 2) Determine if the high steady state level of mRNA encoding the kidney" isoform of vacuolar H+ATPase Mr 56,000 subunit can be accounted for by high levels of transcription alone, or additional posttranscriptional mechanisms, such as mRNA stability. 3) Determine the extent to which the genomic regulatory elements contribute to tissue specific and amplified expression of the "kidney" isoform of the vacuolar H+ATPase Mr 56,000 subunit. 4) Determine the extent to which mRNA stability contributes to tissue specific and amplified expression of the "kidney' isoform of the vacuolar H+ATPase Mr 56,000 subunit. 5) Determine if the expression of the "kidney' isoform of the vacuolar H+ATPase Mr 56,000 subunit is modulated by increased acid intake in the rat, 6) Develop an immortalized intercalated cell line by making mice transgenic for SV40 large T antigen under control of the "kidney isoform promoter.